Phosphatidylinositol is one of a number of phospholipids found in cell membranes, and which participate in intracellular signal transduction. Cell signaling via 3′-phosphorylated phosphoinositides has been implicated in a variety of cellular processes, e.g., malignant transformation, growth factor signaling, inflammation, and immunity (Rameh et al (1999) J. Biol Chem. 274:8347-8350). The enzyme responsible for generating these phosphorylated signaling products, phosphatidylinositol 3-kinase (also referred to as PI 3-kinase or PI3K), was originally identified as an activity associated with viral oncoproteins and growth factor receptor tyrosine kinases that phosphorylate phosphatidylinositol (PI) and its phosphorylated derivatives at the 3′-hydroxyl of the inositol ring (Panayotou et al (1992) Trends Cell Biol 2:358-60). Phosphoinositide 3-kinases (PI3K) are lipid kinases that phosphorylate lipids at the 3-hydroxyl residue of an inositol ring (Whitman et al (1988) Nature, 332:664). The 3-phosphorylated phospholipids (PIP3s) generated by PI3-kinases act as second messengers recruiting kinases with lipid binding domains (including plekstrin homology (PH) regions), such as Akt and PDK1, phosphoinositide-dependent kinase-1 (Vivanco et al (2002) Nature Rev. Cancer 2:489; Phillips et al (1998) Cancer 83:41).
The PI3 kinase family comprises at least 15 different enzymes sub-classified by structural homology and are divided into 3 classes based on sequence homology and the product formed by enzyme catalysis. The class I PI3 kinases are composed of 2 subunits: a 110 kd catalytic subunit and an 85 kd regulatory subunit. The regulatory subunits contain SH2 domains and bind to tyrosine residues phosphorylated by growth factor receptors with a tyrosine kinase activity or oncogene products, thereby inducing the PI3K activity of the p110 catalytic subunit which phosphorylates its lipid substrate. Class I PI3 kinases are involved in important signal transduction events downstream of cytokines, integrins, growth factors and immunoreceptors, which suggests that control of this pathway may lead to important therapeutic effects such as modulating cell proliferation and carcinogenesis. Class I PI3Ks can phosphorylate phosphatidylinositol (PI), phosphatidylinositol-4-phosphate, and phosphatidylinositol-4,5-biphosphate (PIP2) to produce phosphatidylinositol-3-phosphate (PIP), phosphatidylinositol-3,4-biphosphate, and phosphatidylinositol-3,4,5-triphosphate, respectively. Class II PI3Ks phosphorylate PI and phosphatidylinositol-4-phosphate. Class III PI3Ks can only phosphorylate PI. A key PI3-kinase isoform in cancer is the Class I PI3-kinase, p110α as indicated by recurrent oncogenic mutations in p110α (Samuels et al (2004) Science 304:554; U.S. Pat. Nos. 5,824,492; 5,846,824; 6,274,327). Other isoforms may be important in cancer and are also implicated in cardiovascular and immune-inflammatory disease (Workman P (2004) Biochem Soc Trans 32:393-396; Patel et al (2004) Proc. Am. Assoc. of Cancer Res. (Abstract LB-247) 95th Annual Meeting, March 27-31, Orlando, Fla., USA; Ahmadi K and Waterfield M D (2004) “Phosphoinositide 3-Kinase: Function and Mechanisms” Encyclopedia of Biological Chemistry (Lennarz W J, Lane M D eds) Elsevier/Academic Press), Oncogenic mutations of p110 alpha have been found at a significant frequency in colon, breast, brain, liver, ovarian, gastric, lung, and head and neck solid tumors. About 35-40% of hormone receptor positive (HR+) breast cancer tumors harbor a PIK3CA mutation. PTEN abnormalities are found in glioblastoma, melanoma, prostate, endometrial, ovarian, breast, lung, head and neck, hepatocellular, and thyroid cancers.
Upregulation of the phosphoinositide-3 kinase (PI3K)/Akt signaling pathway is a common feature in most cancers (Yuan and Cantley (2008) Oncogene 27:5497-510). Genetic deviations in the pathway have been detected in many human cancers (Osaka et al (2004) Apoptosis 9:667-76) and act primarily to stimulate cell proliferation, migration and survival. Activation of the pathway occurs following activating point mutations or amplifications of the PIK3CA gene encoding the p110a PI3K isoforms (Hennessy et al (2005) Nat. Rev. Drug Discov. 4:988-1004). Genetic deletion or loss of function mutations within the tumor suppressor PTEN, a phosphatase with opposing function to PI3K, also increases PI3K pathway signaling (Zhang and Yu (2010) Clin. Cancer Res. 16:4325-30. These aberrations lead to increased downstream signaling through kinases such as Akt and mTOR and increased activity of the PI3K pathway has been proposed as a hallmark of resistance to cancer treatment (Opel et al (2007) Cancer Res. 67:735-45; Razis et al (2011) Breast Cancer Res. Treat. 128:447-56).
PI3 kinase is a heterodimer consisting of p85 and p110 subunits (Otsu et al (1991) Cell 65:91-104; Hiles et al (1992) Cell 70:419-29). Four distinct Class I PI3Ks have been identified, designated PI3K α (alpha), β (beta), δ (delta), and ω (gamma), each consisting of a distinct 110 kDa catalytic subunit and a regulatory subunit. Three of the catalytic subunits, i.e., p110 alpha, p110 beta and p110 delta, each interact with the same regulatory subunit, p85; whereas p110 gamma interacts with a distinct regulatory subunit, p101. The patterns of expression of each of these PI3Ks in human cells and tissues are distinct. In each of the PI3K alpha, beta, and delta subtypes, the p85 subunit acts to localize PI3 kinase to the plasma membrane by the interaction of its SH2 domain with phosphorylated tyrosine residues (present in an appropriate sequence context) in target proteins (Rameh et al (1995) Cell, 83:821-30; Volinia et al (1992) Oncogene, 7:789-93).
Measuring expression levels of biomarkers (e.g., secreted proteins in plasma) can be an effective means to identify patients and patient populations that will respond to specific therapies including, e.g., treatment with therapeutic agents. There is a need for more effective means for determining which patients with hyperproliferative disorders such as cancer will respond to which treatment with therapeutic agents, and for incorporating such determinations into more effective treatment regimens for patients, whether the therapeutic agents are used as single agents or combined with other agents.
The PI3 kinase/Akt/PTEN pathway is an attractive target for cancer drug development since such agents would be expected to inhibit cellular proliferation, to repress signals from stromal cells that provide for survival and chemoresistance of cancer cells, to reverse the repression of apoptosis and surmount intrinsic resistance of cancer cells to cytotoxic agents. PI3 kinase inhibitors have been reported (Yaguchi et al (2006) Jour. of the Nat. Cancer Inst. 98(8):545-556; U.S. Pat. Nos. 7,173,029; 7,037,915; 6,608,056; 6,608,053; 6,838,457; 6,770,641; 6,653,320; 6,403,588; 7,750,002; WO 2006/046035; U.S. Pat. No. 7,872,003; WO 2007/042806; WO 2007/042810; WO 2004/017950; US 2004/092561; WO 2004/007491; WO 2004/006916; WO 2003/037886; US 2003/149074; WO 2003/035618; WO 2003/034997; US 2003/158212; EP 1417976; US 2004/053946; JP 2001247477; JP 08175990; JP 08176070).
Certain thienopyrimidine compounds have p110 alpha binding, PI3 kinase inhibitory activity, and inhibit the growth of cancer cells (Wallin et al (2011) Mol. Can. Ther. 10(12):2426-2436; Sutherlin et al (2011) Jour. Med. Chem. 54:7579-7587; US 2008/0207611; U.S. Pat. Nos. 7,846,929; 7,781,433; US 2008/0076758; U.S. Pat. No. 7,888,352; US 2008/0269210. GDC-0941 (CAS Reg. No. 957054-30-7, Genentech Inc.), is a selective, orally bioavailable inhibitor of PI3K with promising pharmacokinetic and pharmaceutical properties (Folkes et al (2008) Jour. of Med. Chem. 51(18):5522-5532; U.S. Pat. No. 7,781,433; Belvin et al, American Association for Cancer Research Annual Meeting 2008, 99th:April 15, Abstract 4004; Folkes et al, American Association for Cancer Research Annual Meeting 2008, 99th:April 14, Abstract LB-146; Friedman et al (2008), American Association for Cancer Research Annual Meeting 2008, 99th:April 14, Abstract LB-110) and shows synergistic activity in vitro and in vivo in combination with certain chemotherapeutic agents against solid tumor cell lines (US 2009/0098135).
New targeted treatments that increase treatment responsiveness, delay disease progression, improve tolerability, and potentially eradicate chemotherapy-resistant malignant cells would represent a significant advance in the care of women with breast cancer. The principal strategy for the treatment of patients with hormone receptor positive (HR+) metastatic breast cancer (MBC) has been to block the action of estrogen at the level of the receptor or to reduce estrogen production. Breast cancer is known to be a heterogeneous disease. Different subtypes exist which can be defined based on: (i) the molecular profile of the breast cancer tumor; (ii) genetic array testing; or (iii) approaches using immunohistochemical analyses. Most breast cancers are luminal tumors. Luminal tumor cells look the most like the cells of breast cancers that start in the inner (luminal) cells lining the mammary ducts. Molecular subtypes of breast cancer may be useful in planning treatment and developing new therapies, including: Luminal A, Luminal B, Triple negative/basal-like, and HER2 type. The Luminal A subtype represents about 40% of breast cancer and tends to be ER+ (estrogen receptor-positive) and/or PR+ (progesterone receptor-positive), HER2− (HER2/neu receptor-negative), low Ki67. ER/PR/HER2 are clinically validated predictive and prognostic biomarkers. Proliferation marker Ki-67 is an exemplary prognostic parameter in breast cancer patients.
Aromatase inhibitors (AIs), such as anastrozole, letrozole, and exemestane, block peripheral estrogen synthesis by inhibiting aromatase, the enzyme responsible for the peripheral conversion of androgens to estrogen (Simpson and Dowsett (2002) Recent Prog Horm Res 57:317-38). Clinical trials have demonstrated the efficacy of both tamoxifen and AI therapy in the treatment of postmenopausal women with HR-positive MBC (Nabholtz et al. (2000) J Clin Oncol 18:3758-67; Mouridsen et al. (2001) J Clin Oncol 19:2596-606; Osborne et al. (2002) J Clin Oncol 20:3386-95; Howell et al. (2004) J Clin Oncol 22:1605-13; Paridaens et al. (2004) Proceedings Am Soc Clin Oncol 22:14S). In addition, fulvestrant, an estrogen receptor (ER) antagonist that down-regulates ER protein levels, has been approved by the U.S. Food and Drug Administration (FDA) for HR-positive MBC and European Medicines Agency (EMA) for the treatment of ER-positive MBC in postmenopausal women with disease progression following previous anti-estrogen therapy (Howell et al. (2002) J Clin Oncol 20:3396-403; Osborne et al. (2002) J Clin Oncol 20:3386-95; Di Leo et al. (2010) J Clin Oncol 28:4594-600). Although existing treatments can provide some delay in the progression of disease, the major limitation of endocrine therapy remains the nearly universal development of therapeutic resistance, which leads to eventual death in the overwhelming majority of patients. MBC remains the second highest cause of cancer death in women, emphasizing the continued unmet need in this disease.
Abnormal activation of the phosphoinositide 3-kinase (PI3K) pathway in cancer, either via genetic alterations in PI3K pathway constituents (PI3K-activating mutations or genetic amplification, loss of the antagonistic tumor suppressor PTEN) or via the transduction of aberrant receptor tyrosine kinase (RTK) signals, is a common finding in a variety of tumor types. This combined with the resistance to endocrine therapy suggests that inhibition of PI3K signaling could have broad application in the treatment of breast cancer.
Luminal A breast cancers have higher estrogen receptor (ER+) and/or progesterone receptor (PR+) expression than luminal B breast cancers. Luminal A breast cancer patients with these expression patterns respond well to endocrine, hormone therapy and have a generally favorable prognosis (Rouzier et al (2005) Clin. Cancer Res. 11:5678-5685).
The activity of endocrine therapies, including tamoxifen and AIs is the primary reason for the sustained improvement in survival for patients with early-stage HR-positive breast cancer. However, nearly half of the patients that present with metastatic HR-positive disease do not respond to front-line endocrine treatment and nearly all patients who do respond eventually develop resistance to endocrine therapy.
The mechanisms of resistance to hormonal therapies in HR-positive MBC patients are likely to be multifactorial. Nonclinical and clinical data suggest that decrease or loss of ER and/or PgR expression and upregulation of growth factor signaling are two of the prominent mechanisms leading to estrogen-independent tumor growth (Johnston 2009; Osborne and Schiff 2011). The loss of ER expression over time has been observed in up to 20% of the patients treated with endocrine therapy (Gutierrez et al. 2005), which might account for acquired anti-estrogen resistance and subsequent disease progression. ER expression has been shown to be regulated through multiple growth factor-signaling pathways. In particular, activation of the EGFR/HER2 and mitogen-activated protein kinase (MAPK) pathway leads to suppression of ER expression resulting in resistance to tamoxifen (McClelland et al. 2001; Knowlden et al. 2003; Hutcheson et al. 2003). In addition to modulating ER levels, growth factor-signaling pathways can directly enhance the transcriptional activity of ER via direct phosphorylation of the receptor at serine 118 and serine 167 (Chen et al. 2002; Campbell et al. 2001). “Non-genomic” activities of ER have been postulated to stimulate a number of intracellular signaling pathways including the MAPK and PI3K pathways in the cytosol (Bjornstrom and Sjöberg 2005; Acconcia et al. 2005). These non-genomic activities have been proposed as an important feature in endocrine response and resistance in breast cancer (Schiff et al. 2003).
Multiple lines of nonclinical and clinical data support a key role for the PI3K pathway in the generation of resistance to hormonal therapies. Activation of the PI3K pathway (via PIK3CA mutations, loss of PTEN expression, or HER2 overexpression) has been demonstrated to promote resistance to anti-estrogen therapy and hormonal independence in ER-positive breast cancer models (Shou et al. (2004) J Natl Cancer Inst 96:926-3; Miller et al. (2009) Cancer Res 2009; 69:4192-201, Miller et al. (2010) J Clin Invest 120:2406-13). Proteomic and transcriptional profiling of human HR-positive tumors suggest that increased PI3K signaling is associated with lower ER levels, which has been correlated with resistance to endocrine therapy (Creighton et al. 2010; Miller et al. (2010) J Clin Invest 120:2406-13). Retrospective analyses of tumor samples from HR-positive patients treated with tamoxifen lend support to the nonclinical observations linking the PI3K pathway to resistance to anti-estrogen therapy; patients with an activated PI3K pathway have been found to have decreased overall survival (OS) (Kirkegaard et al. (2005) J Pathol 207:139-46) and shorter relapse-free survival (Shoman et al. (2005) Mod Pathol 18:250-9) Inhibition of the PI3K/mTOR pathway in nonclinical models has been shown to upregulate ER/PgR expression (Creighton et al. 2010) and enhance the antitumor effect of letrozole (Boulay et al. 2005).
In the clinical setting, data from two Phase II studies suggest that the combined inhibition of the PI3K/mTOR and estrogen-signaling pathway may provide superior benefit when compared to single-agent endocrine therapies. Administration of a rapalog, everolimus, increased the efficacy of letrozole in the neoadjuvant setting in patients with ER-positive breast cancer as measured by a decrease in Ki67 expression (Baselga et al. 2009). The addition of everolimus to tamoxifen in a Phase II study with ER-positive patients who received prior treatment with an AI significantly improved the clinical benefit rate (CBR), time to progression and overall survival compared to single-agent tamoxifen (Bachelot et al. 2012). Finally, data from the Phase III BOLERO-2 study demonstrated that the addition of everolimus to exemestane more than doubled PFS compared with single-agent exemestane in ER-positive, HER2-negative MBC patients whose disease was refractory to prior treatment with letrozole or anastrozole (Baselga et al. 2012). Therefore, the combined inhibition of the ER and PI3K-pathways may prove to be an effective therapy in patients with MBC who experience recurrent or progressive disease (PD) while receiving treatment with an AI.
GDC-0941 (pictrelisib, pictilisib, Genentech Inc., Roche, RG-7321, CAS Reg. No. 957054-30-7), named as 4-(2-(1H-indazol-4-yl)-6-((4-(methylsulfonyl)piperazin-1-yl)methyl)thieno[3,2-d]pyrimidin-4-yl)morpholine, has potent PI3K activity (WO 2011/036280; U.S. Pat. Nos. 8,242,104; 8,343,955) and is being studied in patients with locally advanced or metastatic solid tumors. GDC-0941 is a weak inhibitor of Class II, III, and IV PI3K family members (including DNA-dependent protein kinase and mammalian target of rapamycin [mTOR], with >250-fold selectivity for p110α). GDC-0941 potently inhibits in vitro growth of a broad array of breast cancer cell lines by inducing G1 arrest and apoptosis (Friedman et al. (2009) AACR-NCI-EORTC Molecular Targets and Cancer Therapeutics, 15-19 Nov. 2009, Boston, Mass. Abstract C201; O'Brien et al. (2010) Clin Cancer Res 16:3670-83). GDC-0941 has exceeded its minimum effective target dose (an exposure associated with 90% tumor grown inhibition in preclinical models) in Phase I testing, and has demonstrated pharmacodynamic and anti-tumor activity at tolerable doses (Baird et al. (2010) J Clin Oncol 2010 ASCO Annual Meeting Proceedings (Post-Meeting Edition) 2010; 28(15 Suppl):2613; Dolly et al. (2010) J Clin Oncol 2010 ASCO Annual Meeting Proceedings (Post-Meeting Edition) 28(15 Suppl):3079; Von Hoff et al. (2010) J Clin Oncol 2010 ASCO Annual Meeting Proceedings 2010; 28(15 Suppl):2541).